Nickel Carcinogenesis Mechanism: DNA Damage

Nickel (Ni) is known to be a major carcinogenic heavy metal. Occupational and environmental exposure to Ni has been implicated in human lung and nasal cancers. Currently, the molecular mechanisms of Ni carcinogenicity remain unclear, but studies have shown that Ni-caused DNA damage is an important carcinogenic mechanism. Therefore, we conducted a literature search of DNA damage associated with Ni exposure and summarized known Ni-caused DNA damage effects. In vitro and vivo studies demonstrated that Ni can induce DNA damage through direct DNA binding and reactive oxygen species (ROS) stimulation. Ni can also repress the DNA damage repair systems, including direct reversal, nucleotide repair (NER), base excision repair (BER), mismatch repair (MMR), homologous-recombination repair (HR), and nonhomologous end-joining (NHEJ) repair pathways. The repression of DNA repair is through direct enzyme inhibition and the downregulation of DNA repair molecule expression. Up to now, the exact mechanisms of DNA damage caused by Ni and Ni compounds remain unclear. Revealing the mechanisms of DNA damage from Ni exposure may contribute to the development of preventive strategies in Ni carcinogenicity.


Introduction
Nickel (Ni) is the 24 th most abundant element in Earth's crust [1]. On the one hand, at a low concentration, Ni is a nutritionally essential trace element for animals [2][3][4]. Ni is also a required trace element for several enzymes that play critical roles in energy and nitrogen metabolism [5]. On the other hand, at a high concentration, Ni is a toxic element [6][7][8][9]. The unique physical and chemical properties of Ni make it and its compounds suitable materials for many applications widely found in modern industries [1]. The widespread use of Ni increases its concentration in biogeochemical cycles and enhances human exposure to it and its compounds through environmental contamination and occupational exposure [10]. Human exposure to Ni occurs primarily via inhalation, ingestion, and dermal absorption [11]. In humans, Ni has been known to cause liver, kidney, spleen, brain, and tissue damage [12][13][14]. The International Agency for Research on Cancer (IARC) has classified Ni compounds such as nickel sulfate (NiSO 4 ), nickel oxide (NiO), nickel hydroxides, and crystalline nickel as carcinogenic agents to humans (Group 1) [15].

Reactive Oxygen Species (ROS) in Ni-Induced DNA Damage
ROS are a group of short-lived, highly reactive, oxygen-containing molecules [72]. ROS play an important role in cancer development [73]. Excessive ROS attack the DNA, which then results in genomic instability that is a promoter of tumorigenesis [72]. Genomic instability has been suggested to be a major driving force of oncogenesis and can account for genetic diversity in many cancers [72]. It has been shown that oxidative stress is the basic toxicological mechanism of Ni overexposure [33,51,74,75]. Ni and Ni compounds increase ROS accumulation through both a direct increase in ROS generation and an antioxidant-system suppression, which then damages the DNA (as shown in Figure 1).

Reactive Oxygen Species (ROS) in Ni-Induced DNA Damage
ROS are a group of short-lived, highly reactive, oxygen-containing molecules [72]. ROS play an important role in cancer development [73]. Excessive ROS attack the DNA, which then results in genomic instability that is a promoter of tumorigenesis [72]. Genomic instability has been suggested to be a major driving force of oncogenesis and can account for genetic diversity in many cancers [72]. It has been shown that oxidative stress is the basic toxicological mechanism of Ni overexposure [33,51,74,75]. Ni and Ni compounds increase ROS accumulation through both a direct increase in ROS generation and an antioxidant-system suppression, which then damages the DNA (as shown in Figure 1).

ROS-Dependent Ni-Induced DNA Damage
Excessive ROS can directly attack the DNA by oxidizing nucleoside bases, producing modified nucleotides (8-hydroxy-2'-deoxyguanosine,   [100][101][102]. The elevated 8-OHdG level is regarded as an indicator of DNA oxidative damage [103]. It has been suggested that synergistic DNA damage induced by simultaneous exposure of Ni compounds is possibly related to ROS [53,104,105].

ROS-Dependent Ni-Induced DNA Damage
Excessive ROS can directly attack the DNA by oxidizing nucleoside bases, producing modified nucleotides (8-hydroxy-2 -deoxyguanosine, 8-OHdG) [100][101][102]. The elevated 8-OHdG level is regarded as an indicator of DNA oxidative damage [103]. It has been suggested that synergistic DNA damage induced by simultaneous exposure of Ni compounds is possibly related to ROS [53,104,105]. Several in vitro and in vivo studies have demonstrated that Ni and Ni compounds can increase DNA oxidative damage marker levels (8-OHdG) [106][107][108][109][110][111]. In vivo, our previous studies showed that dietary NiCl 2 in excess of 300 mg/kg inhibits the antioxidant system, which leads to an increase in DNA oxidative damage markers, e.g., 8-OHdG contents in the lung and the kidney [56,57]. Liu et al. found that treatment with NiSO 4 (20 mg/kg) for 20 days induced ROS accumulation and increased 8-OHdG levels in mouse livers [107]. Treatment of cultured HeLa cells with Ni 3 S 2 (10 µg/mL) can largely increase 8-OHdG contents, whereas NiO (black), NiO (green), and NiSO 4 do not enhance the production of 8-OHdG [111]. NiCl 2 (0.125, 0.25, and 0.5 mM) treatment for 24 h can also induce mitochondrial DNA (mtDNA) damage, including increased mitochondrial 8-OHdG contents and reduced mtDNA contents and mtDNA transcript levels in Neuro2a cells [112]. In recent decades, several studies have reported that Ni and Ni compounds nanoparticles of can also induce DNA damage [113][114][115][116][117][118]. Mo et al. [113] reported that oxidative DNA damage was significantly upregulated in the lungs of mice after being intratracheally instilled with 50 µg Nano-Ni. In vitro, Abudayyak et al. reported that NiO NPs induced excessive ROS generation and then caused DNA oxidative damage (8-OHdG up-regulation) in NRK-52E kidney epithelial cells and SH-SY5Y neuronal cells [114,115]. It has been also shown that Ni and NiO NPs at higher doses (25 and 50 ug/mL) can induce DNA strand breaks, and an increase in the DNA strand breaks is due to intracellular ROS generation [119].
Through co-treatment with the antioxidant N-acetylcysteine (NAC) and NiNPs, NAC can mitigate NiNPs-induced ROS generation and DNA strand breaks, suggesting the potential mechanism of ROS in DNA damage [120]. Ni acetate can increase intracellular ROS generation and DNA strand breaks in Nrf2 knockdown cells, indicating that ROS play an important role in Ni-induced DNA damage [54].

ROS-Independent Ni-Induced DNA Damage
In contrast to most studies, Kumar et al. demonstrated that NiSO 4 affected DNA replication and damaged DNA but did not induce any detectable ROS production in Escherichia coli, concluding that Ni-induced DNA damage is through an ROS-independent pathway [121]. They also demonstrated that Ni exposure specifically affected DNA polymerization and thereby induced DNA damage. A major reason for the differing results may be related to differences between mammals and bacteria.

Interference of Ni with DNA Damage Repair Systems
After DNA damage occurs, specific pathways are activated to facilitate the identification of the damaged regions and their repair [122]. The DNA repair system contributes to the maintenance of the genetic sequence, the correction of DNA damage, and genomic instability [123,124].
Some studies showed that Ni can inhibit the DNA repair function and promote carcinogenesis [121,[125][126][127][128][129]. Arita et al. reported that 29 DNA repair genes were repressed, and two DNA repair genes were overexpressed in the isolated peripheral blood mononuclear cells (PBMC) in Ni-refinery workers [130]. In addition, Scalon et al. [129] reported that DNA double-strand breaks were significantly higher in the NiCl 2 (250 and 500 µM, 48 h) treatment cells than non-NiCl 2 treatment cells after ionizing radiation (IR) exposure. Likewise, Ni also causes DNA damage repair-system repression, which results in damaged DNA not being removed. The accumulation of DNA lesions facilitates the process of tumorigenesis [131]. At present, an increasing number of studies indicate that DNA repair suppression is a non-ignorable mechanism of oncogenesis [43,132,133].
In general, the DNA repair system can always fix and clear damaged DNA that is induced by normal metabolic activities and environmental factors [134]. Recently, the DNA repair mechanism was found to contain direct reversal, nucleotide repair (NER), base excision repair (BER), mismatch repair (MMR), and double strand break repair including homologous recombination repair (HR) and nonhomologous end joining (NHEJ) repair [135,136], as shown in Figure 2. Next, we review the effect of Ni on every DNA damage-repair pathway (Table 1).

Effect of Ni on Direct Reversal
For reversible DNA damage, our body first uses the direct-reversal repair mechanism to correct damaged bases [149]. The basic means of DNA repair way is direct reversal, which corrects damaged DNA with DNA alkylating agents [149]. O 6 -methylguanine DNA methyltransferase (MGMT) and ALKBH α-ketoglutarate Fe(II) dioxygenases (FeKGDs) are the main direct reversal repair proteins [150].
In nickel sulfide (NiS)-treated or NiS-transformed human 16HBE cells, the DNA repair gene MGMT mRNA and protein-expression levels are significantly reduced, suggesting that the downregulation of MGMT expression levels may be an early event involved in NiS-induced cell transformation [137]. MGMT is unique among DNA repair proteins because it acts alone to remove DNA adducts [151]. MGMT repair can remove both methyl and ethyl adducts, and this reaction is a nonenzymatic (stoichiometric) reaction. Previous studies confirmed that MGMT overexpression can

Effect of Ni on Direct Reversal
For reversible DNA damage, our body first uses the direct-reversal repair mechanism to correct damaged bases [149]. The basic means of DNA repair way is direct reversal, which corrects damaged DNA with DNA alkylating agents [149]. O 6 -methylguanine DNA methyltransferase (MGMT) and ALKBH α-ketoglutarate Fe(II) dioxygenases (FeKGDs) are the main direct reversal repair proteins [150].
In nickel sulfide (NiS)-treated or NiS-transformed human 16HBE cells, the DNA repair gene MGMT mRNA and protein-expression levels are significantly reduced, suggesting that the downregulation of MGMT expression levels may be an early event involved in NiS-induced cell transformation [137]. MGMT is unique among DNA repair proteins because it acts alone to remove DNA adducts [151]. MGMT repair can remove both methyl and ethyl adducts, and this reaction is a nonenzymatic (stoichiometric) reaction. Previous studies confirmed that MGMT overexpression can increase the resistance to cancer. MGMT silencing is also associated with DNA hypermethylation, histone modifications, and DNA methyltransferase 1 (DNMT1) upregulation [137]. However, Iwitzki et al. found that Ni treatment does not affect the MGMT protein levels and only inhibits the MGMT activity in Chinese hamster ovary cells (CHO) cells [138]. These differences are possibly due to the different cell types under study or the differences in duration of Ni exposure.
The other type of direct-reversal repair is performed by ALKBH proteins that are members of a superfamily of FeKGDs [152]. Only four ALKBH family proteins (ALKBH1-3 and FTO) have the ability of DNA alkyl damage removal [153]. Ni can also directly inhibit the DNA alkylation repair enzymes, e.g., ALKBH2 and ALKBH3, by replacing iron at the catalytic site and then reducing the direct reversal of DNA damage [139,140].

Effect of Ni on BER
BER mainly repairs oxidative DNA damage, and it can excise and replace a single damaged nucleotide base [154]. The process of BER has two steps. First, DNA glycosylase finds and cuts the damaged DNA base. Second, the DNA repair proteins repair the damaged site [154]. At least 11 distinct mammalian DNA glycosylases are known, such as 8-oxoguanine DNA glycosylase (OGG1), AP endonuclease (APE1), DNA ligase 1 (LIG1), LIG3, and X-ray repair cross-complementing protein 1 (XRCC1) [122,155].
In a nickel smelting worker's serum, the oxidative DNA damage marker (8-OHdG) levels are significantly increased, and BER DNA glycosylase human OGG1 (hOGG1) is significantly lower than that of nonexposed workers [47]. hOGG1 is a single BER enzyme, specifically recognizing and repairing DNA oxidative damage by removing 7,8-dihydro-8-oxoguanine (8-oxoG). Downregulation of hOGG1 expression is associated with aging, neurodegenerative disorders, and cancer [155,156]. Additionally, Ni at 1 µM can inhibit the activity of formamidopyrimidine-DNA glycosylase (Fpg) and 3-methyladenine-DNA glycosylase II (Alk A), which are involved in DNA excision repair [141]. Fpg is a glycosylase-initiating BER enzyme in E. coli and participates in the first step of the BER to remove specific modified bases from the DNA.

Effect of Ni on NER
The aim of NER is to repair DNA damage that has two or more base-impair sites [157,158]. The process of NER entails damaged-site recognition, damaged-strand removal, and DNA ligation [159][160][161][162]. The NER process needs the participation of many proteins, such as Xeroderma pigmentosum (XP) complementation Groups A through G, excision repair cross-complementation group 1 (ERCC1), and proliferating cell nuclear antigen (PCNA) [163,164].
The NER pathway plays an important role in the prevention of cancer formation, such as bladder cancer and lung cancer [165]. It has been shown that Ni inhibits the removal of UV-induced DNA damage by disturbing DNA repair proteins and affecting the NER process [127,[142][143][144]. Hartwig et al. showed that Ni inhibits NER through the disruption of the incision step in HepG2 and CHO cells [127,145]. The preferential binding of Ni(II) to the protein (DNA repair enzyme) fraction, as compared to DNA and RNA fractions, is the reason for NER incision-step inhibition [146]. Ni also inhibits the NER incision step, possibly through direct interaction with zinc finger repair enzymes such as XPA and poly ADP-ribose polymerase (PARP), and the displacement of the zinc ions [125][126][127][128]131,144,166].
Kim et al. [147] found that treatment with nickel acetate 20 µM for 24 h can induce p53-mediated NER DNA repair-pathway suppression, which is a promoter of tumor development. Nickel acetate inhibits p53 transcriptional activity and then suppresses DNA-damage-inducible protein 45 alpha (GADD45A) expression. Typically, the GADD45A, PCNA, and XPG complex works in the NER incision process, and the function of this complex is site 3 of damage cleavage [167]. Downregulation of GADD45A expression levels results in GADD45A, PCNA, and XPG complex inhibition, which then impairs the GADD45A-mediated NER mechanism [147].

Effect of Ni on MMR
DNA MMR is also a critical pathway for DNA damage. The MMR system maintains genomic stability by repairing base-base mismatches and insertion/deletion loops that arise from DNA replication, thereby preventing mutations from becoming permanent in dividing cells [168,169]. Defects in MMR increase the spontaneous mutation rate and sporadic human cancers [170,171]. MMR is a complex reaction involving multiple proteins that recognize a mismatched base, excise the DNA damage, and resynthesize the DNA sequence containing the correct base and using the parental strand as a template [172]. In general, MMR is initiated when highly conserved proteins (MutS homolog  [173]. In cases of insertion/deletions loops with two or more extra bases, MSH2 and MSH3 are responsible for detection. Following recognition, one of the mutL homolog (MLH) heterodimers binds to the mismatch, and PCNA is loaded onto the DNA by replicating factor C, activating MLH to incise the nascent strand and removing the error in an ATP-dependent manner [174,175]. Then, DNA polymerase synthetizes the new strand followed by nick-sealing [176,177].
Scanlon et al. found that treatment with NiCl 2 250 and 500 µM for 48 h could decrease MMR MLH1 protein and mRNA expression levels in tumorigenic (A549) and nontumorigenic (BEAS-2B) human lung cells [129], and that NiCl 2 could directly bind to the MLH1 gene promoter and then reduce its activity of it. However, it has been demonstrated that there are no alterations of DNA repair genes including hMLH1 and hMSH6 in NiS-transformed 16HBE cells [137].

Effect of Ni on Double-Strand Breaks Repair Pathways
Double-strand breaks (DSB), a DNA damage type, causes changes to the DNA sequence [178]. After DSB occur, repair systems NHEJ or HR are activated [179]. HR, a conservative process, fixes the damaged DNA to be the same as the original DNA sequence. In this process, the damaged DNA sequence is removed, and the new DNA synthesis is according to the homologous sister chromatid [180]. The proteins involved in the HR are shown in Figure 2, including breast cancer 1 (BRCA1), BRCA2, RAD51 (human homolog of Saccharomyces cerevisiae RAD50), fanconi anemia group D2 protein (FANCD2), and partner and localizer of BRCA2 (PALB2) genes [180,181].
In mammalian cells, NHEJ is a preferential way to repair DSB because a homologous template is not necessarily needed in NHEJ rejoins [182]. In NHEJ, the Ku70/Ku80 heterodimer recognizes and binds the two ends of the broken DNA strands [183]. Multiple enzymes are involved in the rejoining process, including LIG4, X-ray repair cross complementing 4 (XRCC4), and DNA-dependent protein kinase (DNA-PK) [183]. LIG4, XRCC4, and DNA-PK heterodimers are recruited by the Ku70/Ku80 heterodimer to the damage site promoting the ligation of the two ends [182].
Takahashi et al. [184] reported that treatment with 40 mM or more of NiCl 2 for 30 min could inhibit the repair of DNA double-strand breaks in Chinese hamster ovary cells. NiCl 2 also inhibited DNA repair only at cytotoxic concentrations at which the cells lost their proliferative ability. Scanlon et al. found that NiCl 2 (250 and 500 µM for 48 h) exposure led to the downregulation of HR without downregulation of NHEJ repair, and that NiCl 2 decreased the HR proteins (BRCA1, RAD51, and FANCD2) and the mRNA expression levels in tumorigenic (A549) and nontumorigenic (BEAS-2B) human lung cells [129]. There were no changes in the NHEJ repair proteins (DNA-PK, KU80, XRCC4, LIG4 protein, and mRNA expression levels) in tumorigenic (A549) and non-tumorigenic (BEAS-2B) human lung cells [129]. The mechanism of the NiCl 2 -inhibited HR pathway is through the transcriptional repression of DNA repair proteins. After evaluating the DSB DNA repair pathway by the ex vivo GFP reporter assay system, Morales et al. found that NiCl 2 at low doses (100 µM) activated the HR pathway but did not affect the NHEJ repair pathway [148]. In contrast, the highest tested dose of NiCl 2 (500 µM) significantly inhibited the HR and the NHEJ repair pathways in U2OS cells [148]. The different results of Morales et al. and Scanlon et al. on the NiCl 2 -affected NHEJ repair pathway may be because of NiCl 2 treatment concentration or the cell model.

Others
When bacterial DNA encounters heavy doses of DNA-damaging agents, it activates the special DNA repair-system SOS response [185]. In general, SOS response repairs the damaged DNA and ensures the process of DNA replication [186]. If the SOS response is suppressed, the evolution of bacterial resistance and pathogens is prevented more easily [187]. Kumar et al. [121] reported that exposure to 1 M Ni ions for 15 h induced double-strand breaks of E. coli. In DNA, meanwhile, the SOS response is impaired by RecBCD function blockage.

Conclusions and Future Perspectives
There have been many studies on the molecular mechanism of Ni and Ni compounds-induced DNA damage associated with carcinogenesis. However, the exact mechanisms of DNA damage caused by Ni and Ni compounds are still unclear. Previous studies have demonstrated that Ni can induce DNA damage, and that Ni-induced DNA damage is mainly through ROS generation. Ni can also directly bind DNA and induce DNA damage. Meanwhile, Ni can also repress the DNA damage-repair systems, including DNA direct-reversal, NER, BER, HDR, MMR, and NHEJ repair pathways, which increases the accumulation of the damaged DNA bases. The repression of DNA repair is through impacting cellular DNA repair on multiple levels, from direct enzyme inhibition to the modulation of DNA repair-molecule expression (Figure 3). Ni exposure can therefore directly induce cancer through DNA damage and DNA damage-repair inhibition. On the other hand, DNA damage-repair inhibition induced by Ni can also increase the risk of other agents (ultraviolet light, ionizing radiation, chemicals, etc.) promoting caner. Ni-induced DNA repair pathway suppression results in damaged DNA accumulation in the cells. If the damaged DNA cell can survive, the damaged DNA is passed down through the damage site to daughter cells, which thus contributes to potential carcinogenesis. ensures the process of DNA replication [186]. If the SOS response is suppressed, the evolution of bacterial resistance and pathogens is prevented more easily [187]. Kumar et al. [121] reported that exposure to 1 M Ni ions for 15 h induced double-strand breaks of E. coli. In DNA, meanwhile, the SOS response is impaired by RecBCD function blockage.

Conclusions and Future Perspectives
There have been many studies on the molecular mechanism of Ni and Ni compounds-induced DNA damage associated with carcinogenesis. However, the exact mechanisms of DNA damage caused by Ni and Ni compounds are still unclear. Previous studies have demonstrated that Ni can induce DNA damage, and that Ni-induced DNA damage is mainly through ROS generation. Ni can also directly bind DNA and induce DNA damage. Meanwhile, Ni can also repress the DNA damagerepair systems, including DNA direct-reversal, NER, BER, HDR, MMR, and NHEJ repair pathways, which increases the accumulation of the damaged DNA bases. The repression of DNA repair is through impacting cellular DNA repair on multiple levels, from direct enzyme inhibition to the modulation of DNA repair-molecule expression (Figure 3). Ni exposure can therefore directly induce cancer through DNA damage and DNA damage-repair inhibition. On the other hand, DNA damagerepair inhibition induced by Ni can also increase the risk of other agents (ultraviolet light, ionizing radiation, chemicals, etc.) promoting caner. Ni-induced DNA repair pathway suppression results in damaged DNA accumulation in the cells. If the damaged DNA cell can survive, the damaged DNA is passed down through the damage site to daughter cells, which thus contributes to potential carcinogenesis. An increasing number of studies confirm that ROS generation is a basis mechanism of Ni toxicity [6]. However, there are no studies about the ROS generation in Ni-induced DNA damage repair inhibition. In the next study, we need more research to explore whether ROS are also essential or just participates in Ni-induced DNA damage-repair pathway inhibition in the carcinogenesis.   An increasing number of studies confirm that ROS generation is a basis mechanism of Ni toxicity [6]. However, there are no studies about the ROS generation in Ni-induced DNA damage repair inhibition. In the next study, we need more research to explore whether ROS are also essential or just participates in Ni-induced DNA damage-repair pathway inhibition in the carcinogenesis.

Conflicts of Interest:
The authors declare that there are no conflicts of interest.